(450a) Aligned Collagen-GAG Scaffolds and Soluble Factor Presentation for Tendon Tissue Engineering

Within the United States there are more than 32 million injuries to tendons and ligaments per year with an associated cost of $30 billion. While small tendon injuries can heal naturally, larger injuries undergo a repair-mediated process that does not recapitulate the native hierarchical structure of tendon extracellular matrix (ECM) and which displays inferior biomechanical properties. Current surgical and tissue engineering approaches have had only limited success, particularly due to high failure rates. Results in the literature suggest that successful regeneration templates for aligned tissues such as tendon must recapitulate aspects of the native tissue anisotropy. Collagen-glycosaminoglycan (CG) scaffolds have previously been used as successful regeneration templates for skin and peripheral nerves, but do not typically have requisite microstructural anisotropy or mechanical integrity for tendon applications. The objective of this study was to develop a CG scaffold with aligned pore microstructure and sufficient mechanical competence for tendon tissue engineering, and to use soluble factors to improve scaffold bioactivity, notably tenocyte (TC) integration and viability.

Composite scaffolds were created from a porous CG scaffold and dense CG membrane. CG membranes were created via an evaporative process. CG scaffolds were fabricated via lyophilization in a manner that integrated the CG membrane; freezing temperature (-60^oC vs. liquid nitrogen ? LN2) and mould thermal conductivity mismatches were used to create aligned pores. Scaffold microstructure was assessed via SEM imaging and stereology. SEM analysis of CG scaffolds shows aligned, elongated pores (aspect ratio 1.5:1) in the longitudinal plane of cylindrical (4 ? 6mm dia, 15 ? 20 mm long) CG scaffold plugs compared to rounder pores (aspect ratio 1:1) in the transverse plane (Fig. 1). A significant affect of CG suspension freezing temperature was observed on scaffold pore size: mean transverse plane pore sizes of 35 μm vs. 55 μm were observed for LN2 vs. -60^oC freezing temperatures. CG scaffold-membrane constructs demonstrated ~8-fold increase in tensile modulus over scaffolds alone (E_tension = 1.6 ± 0.5 MPa, hydrated). In vitro, primary and cultured (P2 ? 3) equine TCs were observed to integrate throughout the CG scaffold variants. Scaffold pore size, alignment, and crosslinking density all showed a significant impact on TC attachment, viability, proliferation, and contractile capacity out to 14 days (Fig. 1); an isotropic scaffold fabricated at -40^oC (100 μm pore size) was used as a control. TC chemotaxis into aligned CG scaffolds also increased significantly in response to PDGF-BB (Fig 1). Ongoing work will further improve construct mechanical competence as well as optimize CG scaffold microstructure (pore size, alignment) and PDGF-BB presentation (soluble vs. insoluble) to speed TC integration, alignment, and functional collagen synthesis in vitro prior to in vivo implantation experiments.

Fig. 1. (top) SEM image and corresponding best-fit ellipse calculated via stereology for longitudinal (left) and transverse (right) CG scaffold sections (-60^oC). (bottom, right) TC attachment and viability increased significantly with time (out to day 14 in vitro), particularly for aligned scaffolds fabricated at -60^oC. (bottom, left) PDGF-BB significantly improved tenocyte chemotaxis (out to day 4) over media alone for all scaffold groups.